PROJECT SUMMARY Protein-peptide interactions are implicated in the etiology of many human diseases and known to play a crucial role in the undertaking of numerous cellular processes. Relatively little is known, however, about the role they play in regulating and fine-tuning carbohydrate signaling. Binding of Insulin-like growth factor-binding protein-3 (IGFBP-3) to the peptide, humanin, is well-studied and so is the interaction of the glycosaminoglycan, hyaluronan (HA), with its main receptor, CD44. However, while it is widely accepted that HA-receptor interaction is necessary for HA-induced effects, much remains to be unveiled about its basic operative mechanisms. The objective of this proposal is to investigate the physical and functional interactions between IGFBP-3 and humanin and their role in mediating HA-CD44 binding and signaling. Knowledge of this interplay and interlinkages involved is currently lacking. Accomplishing our aims in this proposal will provide needed insights into these linkages and will likely lead to novel understanding of the molecular mechanisms underlying fundamental protein- peptide-carbohydrate interactions. Our central hypothesis is that IGFBP-3 binds to HA and blocks its interaction with CD44, inhibiting cell survival. Humanin counteracts this effect by binding to IGFBP-3 thus disabling its binding to HA which is now free to interact with CD44. This hypothesis is based on the following observations: 1) humanin is known to bind residues 215-232 of mature IGFBP-3 in the C-terminal region of the protein. 2) This region of IGFBP-3 was shown earlier to bind certain glycosaminoglycans including HA. 3) Our recently published data showing that a) HA binds with a weaker affinity to this region of the protein than does humanin, b) either HA or humanin could bind to this IGFBP-3 segment, but not simultaneously, c) CD44 blocked HA binding to IGFBP-3, d) upon incubation of HA with CD44 and either IGFBP-3 protein or peptide, humanin was effective at binding and sequestering IGFBP-3 or peptide, thereby enabling access of CD44 to HA, e) while IGFBP-3 and humanin in the conditioned media can immuneprecipitate in a complex, the fraction of IGFBP-3 able to bind HA was not complexed with humanin, and f) IGFBP-3 exerts its cytotoxic effects on cell survival through a mechanism that depends on HA-CD44 interactions. Based on these observations, our specific aims are to: 1. Test the hypothesis that binding of IGFBP-3 to HA is inhibited by CD44 N-glycosylation but unaffected by CD44 reduction. 2. Test the hypothesis that there is a positive correlation between cell viability and the amount of IGFBP-3 bound to humanin in CD44-positive cell lines. 3. Test the hypothesis that while both IGFBP-3 and its peptide can disrupt HA-CD44 signaling, IGFBP-3, but not its peptide, can operate in an IGF-IR-dependent manner. Better understanding of these basic mechanisms will likely advance our knowledge of diseases resulting from dysregulation of protein-peptide-carbohydrate signaling. This R15 application provides an effective vehicle for introducing undergraduate and graduate students to an authentic and extensive hands-on research training at an early stage of their education and cultivates an interest in a career in biomedical research.